Process for preparing lysine containing peptides

ABSTRACT

A PROCESS FOR PREPARING PEPTIDES CONTAINING LYSINE WHEREIN THE TERMINAL AMINO GROUP OF LYSINE IS PROTECTED BY A PYRIDYL -4- METHYLOXYCARBONYL GROUP DURING FORMATION OF THE PEPTIDE AND REMOVING THIS GROUP WITH ZINC IN ACID.

United States Patent C Int. Cl. C07c 103/52; C07g 7/00; C08h 1/00 US.Cl. 260-1125 4 Claims ABSTRACT OF THE DISCLOSURE A process for preparingpeptides containing lysine wherein the terminal amino group of lysine isprotected 'by a pyridyl 4 methyloxycarbonyl group during formation ofthe peptide and removing this group with zinc in acid.

The present invention relates to a process for protecting amino groupsduring peptide synthesis. More particularly, this invention relates to aprocess for preparing peptides containing lysine wherein the terminalamino group is protected by a pyridyl 4 methyloxycarbonyl group duringthe peptide condensation reactions and is selectively removed by zinc inacid when the desired peptide product is obtained.

The synthesis of peptides, particularly heteropeptides, is a problemwhich has long challenged the art. Such products are useful in thesynthesis of proteins. Some of them are therapeutically active. They arealso useful in the study and analysis of proteins, especially in studiesdesigned to gain insight into the mode of action of enzymes, hormones,and other proteins with important functions in the body.

The synthesis of peptides containing lysine has presented an especiallydifficult problem because of the 7 presence of an additional terminalamino group which can cause side-reactions to occur. -In most instances,the terminal amino group is prevented from entering into the peptideforming reaction by protecting it with a blocking group which is removedwhen the desired peptide is obtained. Several protecting groups areknown in the art which are useful in the synthesis of peptidescontaining a lysine residue, but many of the known protecting groups areremoved with great difficulty during the final stages of the synthesisor are unstable to some of the reagents generally employed in peptidesynthesis.

A requirement of the protecting group for the terminal aminofunctionality of lysine in peptide synthesis is that it must be stableto the acidic reaction conditions generally employed in removingblocking groups, such as t-butyloxycarbonyl, from other portions of thesynthesized peptide. The t-butyloxycarbonyl group may be removed underacidic conditions, for example, by treatment in trifluoroacetic acid ortreatment with hydrogen chloride in anhydrous or aqueous medium. Aneffective protecting group for the terminal amino group of lysine shouldnot only be stable under these acidic reaction conditions but alsoshould be completely removed without affecting other portions of thepeptide. Commonly applied blocking groups for the terminal'aminofunctionality of lysine include benzyloxycarbonyl, trifluoroacetyl, andp-toluenesulfonyl. The benzyloxycarbonyl blocking group is notcompletely stable under the conditions used to remove thet-butyloxycarbonyl blocking group, and, as a result, a partial loss ofthe terminal benzyloxycarbonyl group occurs whenever one attempts toselectively remove a tbutyloxycarbonyl blocking group in a peptidesynthesis. Removal of the benzyloxycarbonyl protecting group from lysinecontaining peptide can be accomplished by hydrogenation in aheterogeneous system with a Noble metal 3,780,0lfi' Patented Dec. 18,1973 catalyst. This procedure is satisfactory for small peptides,however, large peptides have a highly convoluted or folded tertiarystructure such that the benzyloxycarbonyl group to be removed is ofteninaccessible to the catalyst. Moreover, removal of the benzyloxycarbonylgroups by catalytic hydrogenation becomes unsatisfactory not only whenthe molecule becomes too large, but in the case of sulfur containingpeptides, such as peptides containing methionine or cysteine, theprocedure fails even with relatively small molecules.

The trifluoroacetyl blocking group can also be employed to protect theterminal amino functionality of lysine. Removal of this blocking groupis accomplished under weakly basic conditions, for example in aqueouspiperidine. The disadvantage of this blocking group is that many peptidecondensation reactions are run in Weakly basic media, and, as a result,a loss of the trifluoroacetyl blocking group occurs during peptidesynthesis leading to undesirable side reactions.

The terminal functionality of lysine has also been protected by thep-toluenesulfonyl blocking group. Although this group is stable to theacidic conditions required for the removal of the t-butyloxycarbonyl andbenzyloxycarbonyl blocking groups, eventual cleavage of thep-toluenesulfonyl group is achieved only through the use of sodium inliquid ammonia; these conditions lead to unwanted side reactions Withmany peptides.

The pyridyl 3 methyloxycarbonyl blocking group has been employed toprotect the terminal amino group of lysine. This blocking group can beremoved from peptides by catalytic hydrogenation or by sodium in liquidammonia. Removal of this protecting group by catalytic hydrogenation issatisfactory for small peptides, however, this method is not suitablefor large peptides or with sulfur containing peptides. The disadvantageof using sodium in liquid ammonia, as noted above, is that undesirableside reactions accompany removal of the pyridyl-3-methyloxycarbonylblocking group.

The pyridyl-4-methyloxycarbonyl blocking group for the terminal aminogroup of lysine used according to the process of the present inventionhas distinct advantages over the above-mentioned blocking groups. Theblocking group is stable under acidic or basic conditions from pH 0-12,for example it is possible to remove even the benzyloxycarbonyl blockinggroup selectively in the presence of pyridyl 4 methyloxycarbonyl as forexample with anhydrous hydrogen fluoride. The blocking group is alsostable in aqueous or anhydrous hydrochloric acid at room temperature. Inaddition to the stability of the pyridyl 4 methyloxycarbonyl blockinggroup to the reaction conditions employed in peptide synthesis, thisgroup can, nevertheless, be completely removed under reductiveconditions, for example by zinc in the presence of acid, withouteffecting other portions of the peptide molecule.

According to the process of this invention, the pyridyl-4-methyloxycarbonyl blocking group is introduced into the terminal aminogroup of the lysine by reacting a lysine heavy metal complex withpyridyl-4-methylsuccinimide carbonate.

Pyridyl-4-methylsuccinimide carbonate is prepared by reactingsuccinimidochloroforrnate, prepared according to procedures known in the'art, with 4-pyridylcarbinol. The reaction is carried out by adding thesuccinimidochloroformate dissolved in an organic solvent to an organicsolution of the 4-pyridylcarbinol. The reaction is conducted in asuitable solvent, such as methylene chloride, chloroform, ethyl acetate,and the like. The reaction is generally run at a low temperature,preferably between 20 to 0 C. The 4-pyridylcarbinol is added at a slowrate with stirring in order to prevent the temperature of the reactionfrom going above C. The amount of succinimidochloroformate employed inthis reaction should be slightly more than an equimolar amount of4-pyridylcarbinol used; preferably from 1% to 10% molar excess ofsuccinimidochloroformate is employed. After the addition of thepyridylcarbinol is completed, a tertiary organic amine, for exampleN-methylmorpholine, triethylamine or pyridine, is added at 0 C. Thereaction is allowed to stir at a temperature of from 0 to 5 C. and thenextracted successively with an acid, for example sulfuric acid, anaqueous base, for example sodium bicarbonate, and then water. Thepyridyl-4-methylsuccinimide carbonate is separated and purified bymethods known in the art.

The metal complex of lysine employed in this process is prepared byreacting one mole equivalent of lysine with approximately two moleequivalents of a metal salt in an aqueous medium. The metal salt isselected from the group consisting of copper, cobalt, nickel, aluminum,and the like. The lysine metal complex is obtained by mixing thereagents in an aqueous medium and can be used in the next step of theprocess without further separation or purification.

The preparation of N-e-pyridyl-4-methyloxycarbonyl lysine isaccomplished by reacting a metal complex of lysine withpyridyl-4-methylsuccinimidocarbonate in an aqueous medium. The reactionis carried out under basic conditions, for example at a pH of from 7 to12 and preferably at a pH of 10. The pH of the reaction medium isadjusted to pH 10 by addition of an aqueous base, for example 50% sodiumhydroxide. The pyridyl-4-methylsuccinimidocarbonate is slowly addedwhile the pH is maintained at 9.5 by addition of base. The addition ismade at a temperature of from 040 C. and preferably at 20 C. When theconsumption of base has ceased, the pH is adjusted to 7.5 by addition ofan acid, for example acetic acid, to precipitate the metal complex ofpyridyl-4-methyloxycarbonyllysine which can be separated by filtration.

The free pyridyl-4-methyloxycarbonyllysine can be obtained by treatingthe metal salt with gaseous hydrogen sulfide in water or in aqueousacid, for example 10% acetic acid. The product can be recovered from thereaction mixture by methods known in the art, for example by evaporationof the solvent and crystallization.

Epsilon pyridyl 4 methyloxycarbonyl-u-protected lysine compounds can beprepared by reacting an a-protected lysine withpyridyl-4-methylsuccinimidocarbonate under basic conditions, forexample, pH 7 to 12 and preferably at pH 9.5. An equimolar amount ofpyridyl- 4 methyloxycarbonylsuccinimidocarbonate is slowly added to abasic aqueous solution of the Ot-PI'OtGC-tfid lysine compound at atemperature of from 0-35 C. The pH of the reaction is maintained byaddition of aqueous base, for example, aqueous sodium hydroxide. Afterconsumption of base ceases, the reaction mixture is extracted with anorganic solvent, for example, ethyl acetate, chloroform, methylenechloride, and the like. The pH of the solution is adjusted to 4.2 byaddition of an acid, for example sulfuric acid, and the acidifiedsolution extracted again with the same solvent as above. The organicextracts obtained from the acidified reaction mixture are combined,dried, and evaporated to dryness to afford thee-pyridyl-4-methyloxycarbonyl 0c protected-lysine compound. Purificationof the compounds can be accomplished by conventional techniques, such asrecrystallization and chromatography. Alpha substituted lysine compoundswhich can be employed in the above process includebenzyloxycarbonyllysine, p-nitrobenzyloxycarbonyllysine, pbromobenzyloxycarbonyllysine, p methoxybenzyloxycarbonyllysine,t-butyloxycarbonyllysine, o-nitrophenylsulfenyllysine, 2 (pdiphenyl)-isopropyloxycarbonyllysine, and the like.

The pyridyl-4-methyloxycarbonyl blocking group can be completely removedfrom lysine or a peptide containing a lysine residue by reaction withzinc dust in an aqueous acidic medium. The deblocking reaction iscarried out by dissolving the N-e-pyridyl-4-methyloxycarbonyllysinecompound in 50% aqueous acetic acid, and then adding the zinc dust tothe solution in one portion with high speed stirring. The reaction isconveniently carried out at room temperature, although temperaturesother than room temperature may also be employed. The time the reactionis allowed to stir depends on the nature of the lysine containingcompound or peptide and can vary from 1 to 3 hours. The amount of zincdust employed in the deblocking reaction can vary from 10 to 200 partsper part of pyridyl-4-methyloxyearbonyllysine compound. Complete removalof the blocking group is determined by thin layer chromatography. Thedeblocked peptide can be isolated by methods well known in the art, forexample by gel filtration.

For preparing peptides according to the present invention, thecondensation methods usual in peptide chemistry may be used, such as thecarbodiimide or the azide method, or, for example, the method of mixedanhydrides or of activated esters. The peptides are built up from aminoacids by condensing members selected from the group consisting ofnaturally occurring a-amino acids, peptides built up from said aminoacids, and derivatives thereof, and wherein at least one component ofsaid members is lysine. More particularly, the claimed improvementcomprises protecting the epsilon-amino functionality of the lysineradical with the pyridyl-4-methyloxycarbony1 group.

The term naturally occurring amino acids used herein is to be understoodas referring to all naturally occurring amino acids in their L- orD-form, for example, alanine, arginine, aspartic acid, cysteine,cystine, glutamic acid, glycine, histidine, hydroxylysine,hydroxyproline, isoleucine, leucine, methionine, ornithine,phenylalanine, proline, serine, threonine, tyrosine, and valine.

An example of the use of the process of this invention in peptidesynthesis is the preparation of the nonapeptide T27phenylalanyl-seryl-tryptophanyl glycylalanyl-glutamyl-glycyl-glutaminyl-lysine (hereinafter designated Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys) which exhibits a high degree ofencephalitogenic activity. Experimental allergic encephalomyelitis (EAE)is an experimentally produced inflammatory and demyelinating disease ofthe central nervous system, and a study of the encephalitogenic activityof peptide T27 provides a useful model for the study of autoimmunedisease. The T27 peptide can be prepared by block synthesis, wherein twopeptide segments of the nonapeptide are individually synthesized andthese segments are then coupled in proper sequence to form the desiredpeptide product. The two peptide segments are the tetrapeptide:glutamyl-glycyl-glutaminyl- (N-e-pyridyl- 4-methyloxycarbonyl)lysine andthe pentapeptide; t-butyloxycarbonylphenylalanyl seryl tryptophanylglycylalanine hydrazide. The lysine containing tetrapeptide is preparedin a stepwise coupling procedure by reacting N-e-pyridyl-4-methyloxycarbonyllysine sequentially with N-carboxy-glutamine anhydride, N-thiocarboxy anhydride of glycine andN-carboxy glutamic anhydride according to the methods known in the art.The reaction is conveniently carried out by vigorously agitating thereactants together in aqueous solution at 0 C. at a pH 10.2. TheN-carboxyanhydrides of glutamine and glutamic acid and the N-thiocarboxy anhydride of glycine, prepared by methods known in the art,are added generally all at once or over a period of 15-30 seconds.Alkali, for example potassium hydroxide, is added as required tomaintain the pH. The reaction of the N-thiocarboxy anhydride of glycineis carried out at a pH of 8.85. The reactions are generally completedWithin less than 5 minutes as judged by the cessation of baserequirements. When the reaction is completed, the pH is adjusted to 3 byaddition of a mineral acid, for example hydrochloric acid and the systemis flushed with nitrogen to remove carbon dioxide. The tetrapeptide isisolated by freeze-drying the reaction product and purified by gelfiltration, chromatography and electrophoresis.

The pentapeptide segment is prepared by the Merrifield solid phaseprocedure starting with t-Boc-alanine. In this procedure, the carboxylend of alanine (and of the polypeptide product in the following steps),is bound covalently to an insoluble polymeric resin support, as forexample as the carboxylic ester of the resin-bonded benzyl alcoholpresent in hydroxymethyl-substituted polystyrenedivinylbenzene resin.The t-butyloxycarbonyl protecting group is selectively removed bytreatment of the protected aminoacyl resin with anhydrous hydrogenchloride in an organic solvent, for example dioxane. The aminoacyl resinhydrochloride which results from this treatment is then treated with asolution of triethylamine in chloroform to neutralize the hydrochlorideand liberate the free amine group in a condition ready for coupling withthe next amino acid. The t-butyloxycarbonyl derivative of the next aminoacid desired in the peptide chain is then added, along withdicyclohexylcarbodiimide as the coupling agent. The t-butyloxycarbonylderivatives of the following amino acidsglycine, tryptophane, serine,and phenylalanine are added sequentially. Cleavage of the pentapeptidefrom the resin by treatment with hydrazine in dimethylformamide producesthe pentapeptide hydrazide.

The pentapeptide azide is prepared by treating the t-Boc-Phe-Ser-Trp-Gly-Ala hydrazide in dimethylformamide with isoamylnitritein the presence of hydrochloric acid. The reaction is run between -25 to-35 C. The azide coupling reaction is carried out by adding thetetrapeptide- Glu-Gly-Gln-(e-i-Noc)-Lysdissolved in dimethylformamide tothe above azide solution at a pH of 5 at C. The pH of the reactionmixture is adjusted to 7.2 by addition of diisopropylethylamine and thereaction is stirred at -10 C. for hours to afford a clear thick gelwhich produces a precipitate on addition of ether. The solid isseparated and washed with ether, methanol-water, and Water. The productis obtained by freeze-drying the water washes and drying the residualsolid in vacuo.

The tert-butyloxycarbonyl blocking group is removed from the nonapeptideby treatment with trifluoroacetic acid containing mercaptoethanol as acation scavenger to protect the Trp nucleus. The reaction is run at C.for approximately five minutes and the nonapeptide precipitates onaddition of ether and petroleum ether. The peptide is isolated byremoving the solvents and purified by chromatography. Removal ofpyridyl-4-methyloxycarbonyl blocking group from the nonapeptide isaccomplished by treating the peptide with zinc dust in aqueous aceticacid. The reaction is carried out at 25 C. for One hour. The zinc isremoved from the reaction by centrifugation and the nonapeptide ispurified by chromatography.

The following examples illustrate the invention, but they are notintended to limit it thereto. The abbreviated designations, which areused herein for the amino acid components, their derivatives and certainpreferred protecting groups employed in this invention are as follows:

6 EXAMPLE I (A) Preparation of epsilon-pyridyl=4-methyloxycarbonyllysine(1) Pyridyl 4 methyl-succinimidocarbonate.-4-pyridylcarbinol (6.54 g.,0.06 mole) is dried by distilling off two 75 ml. portions of benzene andis dissolved in 45 ml. of methylene chloride. The solution is cooled to0 and a solution of succinimidochloroformate (11.75 g., 0.065 mole)prepared according to the procedure described in D. Stevenson and G. T.Young, J. Chem. Soc. (c) 2389 (196 9), in 60 m1. of methylene chlorideis added dropwise with stirring at a rate slow enough to prevent heatingabove 0. When the addition is complete a solution of N-methylmorpholine(5.88 g., 0.058 mole) in 8 ml. of methylene chloride is added over aperiod of about two minutes at 0 with stirring. After stirring for 15minutes at 0-5" the solution is rapidly extracted with 150 ml. portionsof .1 N sulfuric acid, saturated sodium bicarbonate, and water. Theaqueous phases are extracted with 40 ml. of methylene chloride and thecombined extracts are dried over sodium sulfate. The methylene chloridesolution is evaporated in vacuo, flushed once with ether and trituratedwith ether. Filtration gave 10.4 g. of crudepyridyl-4-rnethylsuccinimidocarbonate. This material is dissolved in aminimum of ethyl acetate and the solution filtered. 'Hexane is added tothe cloud point and the solution is again filtered. The procedure iscarried out four times. After the final filtration, the solution is alight yellow color. On seeding, the pyridyl-4-methylsuccinimidocarbonatecrystallizes slowly overnight: I.R. shows C=O at 4.59, 5.59, and 7.75;,M.P.9294.

(2) e-Pyridyl-4-methyloxycarbonyllysine.A solution of 1.82 g. (.01 mole)lysine hydrochloride in 10 ml. of water is mixed with a solution of .935g. (.005 mole) CuCl .2H O in 10 ml. of water and an additional 10 ml. ofwater is added. The solution is adjusted to pH 10 with 50% sodiumhydroxide. Pyridyl-4-methylsuccinimidocarbonate (2.50 g., .01 mole) isadded slowly with stirring and the pH maintained at 9.5 by the additionof 50% sodium hydroxide. When base consumption ceases, the pH isadjusted to 7.5 with acetic acid and the precipitated copper complex ofpyridyl-4-methyloxycarbonyllysine is isolated by filtration. Thisprecipitate is suspended in ml. of 10% acetic acid and hydrogen sulfideadded to precipitate Cu(l'iI). The solution is filtered through Celiteand is evaporated to dryness. The resulting oil is crystallized fromWater-ethanol to afford e-N-p'yridyl-4-methyloxycarbonyllysine, M.P.240242 C.

Removal of the blocking group from e-pyridyl-4-methyloxycarbonyllysineis accomplished in the following manner.

10 mg. of N-e-pyridyl-4-methyloxycarbonyllysine is dissolved in 1 ml. of50% aqueous acetic acid and 100 mg. of zinc dust is added. Afterstirring for 1.5 hours at 25 C., the reaction mixture is analyzed bythin layer chromatography and complete conversion to lysine is observed.

EXAMPLE H 'N-a-t-butyloxycarbonyl-N-e-pyridyl-4- methyloxycarbonyllysinea-Butyloxycarbonyllysine acetate (.918 g., .003 mole) is dissolved in 30ml. of Water and the pH adjusted to 9.5 with N sodium hydroxide.Pyridyl-4-methylsuccinimidocarbonate (.750 g., .003 mole) is addedslowly and the pH held at 9.5 by the addition of N sodium hydroxide.When base consumption ceases, the solution is extracted 3 times with 75ml. portions of ethyl acetate, the pH is adjusted to 4.2 by the additionof 2.5 N sulfuric acid. The solution is extracted 4 times with 75 ml.portions of ethyl acetate. The organic extracts at pH 4.2 are combined,dried over sodium sulfate, and evaporated to dryness. Crystallizationfrom ethyl acetate-ethyl ether gives 0.76 g. ofN-a-t-butyloxycarbonyl-N-e-pyridyl-4- 7 methyloxycarbonyllysine, M.P.69.571.5 C.; LR. shows N=H 3.03, C= 5.85, 5.96;.

The removal of the blocking group fromN-rx-tert-butyloxycarbony1-N-e-pyridyl-4-methyloxycarbonyllysine isaccomplished in the following manner.

10 mg. of N a tert butyloxycarbonyl-N-e-pyridyl-4- methyloxycarbonyllysine is dissolved in 1 ml. of 50% aqueous acetic acid and 100 mg.of zinc dust is added. After stirring for four hours at 25 C., analysisby thin layer chromatography indicates complete conversion toN-a-tert-butyloxycarbonyllysine.

When N-a-tert-butyloxycarbonyl-N-e-pyridyl-3-methyloxycarbonyllysine,prepared by the method described above, is employed in the deblockingprocess in place of N-a-tert-butyloxycarbonyl-N-e-pyridyl 4meth'yloxycarbonyllysine and the reaction is stirred for 24 hours, onlyabout 5% of the e-pyridyl-3-methyloxycarbony1 blocking group is removedas shown by thin layer chromatography.

EXAMPLE III Preparation ofphenylalanyl-seryl-tryptophanyl-glycylalanyl-glutamyl-glycyl-glutaminyl-lysine(A) Preparation of glutamyl-glycyl-glutaminyl(N-epyridyl-4-methyloxycarbonyl) lysine.The N-e-pyridyl-4-methyloxycarbonyllysine, 421.7 mg. (1.5 mmoles) and 20 ml. ofpotassium borate buffer (pH 10.2) are placed in a Waring Blender at 0 C.To this is added .276 mg. (1.61 mmoles; 7% molar excess) of crystallineN-carboxy anhydride of glutamine in one portion with rapid stirring. ThepH is maintained at 10.2 by addition of 0.55 ml. of 5 N potassiumhydroxide. The reaction is complete in about two minutes and the pH isadjusted to 3 by addition of concentrated hydrochloric acid; the systemis flushed with nitrogen to remove carbon dioxide. The pH of thereaction mixture is then raised to 8.85 by addition of 5 N potassiumhydroxide and 197 mg. (1.675 mmoles; 5% molar excess) of N-thiocarboxyanhydride of glycine is added in one portion. The addition of 0.47 ml.of 5 N potassium hydroxide requires 1.5 minutes and the reaction isstirred for 5 minutes. The pH is adjusted to 3 by addition ofconcentrated hydrochloric acid and the system is flushed with nitrogento remove carbon oxysulfide. The pH of the reaction mixture is raised to10.1 by addition of 5 N potassium hydroxide and 300 mg. (1.74 mmoles, 3%molar excess) of solid N-carboxy glutamic acid anhydride is added in oneportion. The pH of the reaction is maintained by addition of 0.65 ml. of5 N potassium hydroxide and the reaction is stirred at pH 10.1 for 1.75minutes. The pH is adjusted to 3 by addition of concentratedhydrochloric acid and the reaction product freeze dried to afford 7.09g. This crude freeze dried material is extracted with methanol (oncewith 100 ml. and four times with 50 ml.) and the extracts concentratedin vacuo to afford 1.27 g. of Glu-Gly-Gln- (e-i-Noc)-Lys. The extractedtetrapeptide is purified in the following manner: (a) chromatographedtwice on Sephadex G50 fine in 50% acetic acid; (b) chromatographed onSilica gel column, using chloroformzmethanol:water (60:40:10) as eluent;and (c) preparative electrophoresis in pyridineacetate buffer at pH 6.4.

(B) Preparation oft-butyloxycarbonyl-phenylalanylseryl-triyptophanyl-glycyl-alaninehydrazide.The pentapeptide hydrazide is prepared by solid phase peptidesynthesis in the following manner:

One gram (.77 mmoles) t-Boc-Ala-Resin is suspended in 30 ml. ofmethylene chloride at room temperature. The amino acid resin is treatedas follows: (a) wash 3 times with 15 ml. of dioxane; (b) remove theterminal t-Boc group by reacting 15 ml. of 4 N hydrochloric acid indioxane; (c) wash the deblocked material 3 times with 15 ml. dioxane;(d) wash 3 times with 15 ml. of chloroform; (e) neutralize the materialwith 15 m1. of a mixture of triethylamine in chloroform (1:9); (f) washthe neutralized material 3 times with 15 ml. chloroform;

- (g) wash 3 times with 15 ml. methylene chloride; (h)

add 338 mg. of t-Boc-Gly in methylene chloride; (i) couple by adding397.6 mg. of N,N-dicyclohexylcarbodiimide in methylene chloride andallowing the reaction to run for two hours; (j) wash thet-Boc-Gly-Ala-Resin 3 times with 15 m1. of methylene chloride. Byrepeating these operations with the following amino acid derivatives,t-Boc-Trp (586 mg), t-Boc-Ser (396 mg), t-Boc-Phe (510 ing.), thepentapeptide resin, t-Boc-Phe-Ser-Trp-Gly- Ala-Resin, is prepared. Aftercoupling of t-Boc-Trp, the deblocking which hydrochloric acid is carriedout in the presence of mercaptoethanol to avoid alkylation of Trp. Thepeptide-Resin is washed 3 times with 15 m1. ethanol, 3 times with 15 ml.acetic acid, 3 times with 15 ml. ethanol, and 5 times with 15 ml.methylene chloride and dried in vacuo to afford 1.3 gm. oft-Boc-Phe-Ser- Trp-Gly-Ala-Resin.

1.3 g. of t-Boc-Phe-Ser-Trp-Gly-Ala-Resin is treated with 12 ml. of a1:1 mixture of hydrazine in dimethylformamide for two hours. The resinis collected by filtratration and washed two times with 10 ml. ofdimethylformamide. The dimethylformamide washes and filtrate arecombined and the solvents are removed in vacuo to produce an oilyresidue. The product crystallizes on standing. The solid is washed fivetimes with 20 ml. of ethyl acetate and dried in vacuo to remove tracesof ethylacetate. The dried solid is washed with 15 ml. portions of wateruntil all hydrazine and dimethylformamide impurities are removed. 378.1mg. of t-Boc-Phe-Ser-TrpGly- Ala-hydrazide is obtained.

(C) Azide coupling of peptide fragments A and B.-t-Boc-Phe-Ser-Trp-Gly-Ala-NHNH (144.5 mg., 0.210 mm.) is suspended in1.5 ml. of dimethylformamide and the mixture is cooled to -40. When0.150 ml. of HCl in tetrahydrofuran is added, a. clear solution isobtained, having a pH of 1. The solution is maintained at --25 to 3-0and a total of 33 of isoamylnitrite, 116% of theory, is added inportions during an 18 min. period until a faintly positive starch-iodidetest is constant for 12 minutes. TLC in chloroform-methanol-water (:15:1.5) on silica indicates that all of the hydrazide has been convertedinto Tollens-negative material. An essentially single spot azide isdetected by hypochlorite-starchiodide spray.

Glu-Gly-Gln-(e-i-Noc)-Lys mg., 0.252 mm., 20% excess) is dissolved in0.8 ml. of dimethylformamide and added to the azide solution which hasbeen adjusted to pH 5 with 0.15 ml. of diisopropylethylamine. The tubecontaining the nucleophile is washed out with 2x 0.2 ml. and 1x 0.1 ml.of dimethylformamide. The reaction mixture is adjusted to pH 7.2 byaddition of 100k of diisopropylethylamine. A total of 8 equivalents ofamine is used after 1 minutes at l0, TLC in chloroform-methanol-water(60:40:10) and chloroform-methanol-water-amm'onia (60:30:426) shows thatthe reaction is almost complete, using Ehrlich, ninhydrin, andhypochlorite spray for detection of products. After the reaction mixturehas been stirred magnetically at 1-0 for 20 hours, it is a clear thickgel and addition of 20 ml. of ether gives a precipitate.

The mixture is kept at 0 until a clear supernatant liquid is obtained.The precipitate is removed by centrifugation and washed with 10 ml. ofether, 2X 10% methanolether, and 2X 10 ml. of water. By freeze-dryingthe water washes, 106 mg. of product is obtained. The residual solidafter being dried in vacuo weighs mg.

Gel filtration is used to purify the water washes. The 106 mg. isdissolved in 4 ml. of 50% acetic acid and charged on Sephadex G-50,fine, column.

Solvents are removed in vacuo and the residue is freezedried to t BocPhe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-(E-i- Noc)-Lys.

Amino acid analysis after 20 hr. acid hydrolysis:

1.01 1.0o 'Po.'15 Y2.oo 1 .oo rea mo M.W. 1475 found.

9 Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-(e-i-Noc)-Lys A 94 mg. sample of thewashed solid above is mixed with 1.5 ml. of trifluoroacetic acid,containing 1% mercaptoethanol as a cation scavenger to protect the Trpnucleus. In 3 min. at 25 the solid dissolves completely, and thesolution is kept an additional 2.5 min. at 25. The deblocked peptide isprecipitated with 10 ml. of cold ether and 10 ml. of cold petroleumether and washed with 2X 5 ml. of ether. After being dried in vacuo, thecrude peptide weighs 109 mg.

For purification, the product is dissolved in 6 ml. of 50% acetic acidwith cooling in an ice bath. It is slowly soluble, requiring 30 min. forcomplete dissolution. The solution is charged into a 2.5 X 100 cm.column of Sephadex G-50, fine, and washed out with 2x 1 ml. of 50%acetic acid.

6.2 ml. fractions are collected every 20 min. Fractions 64-72 arecombined, concentrated in vacuo and freezedried giving 78.50 mg. ofPhe-Ser-Trp-Gly-Ala-Glu-Gly- Glne-i-NOC) -Lys.

Amino acid analysis after 20 hr. acid hydrolysis: oss 'i.oo pom z.oo1.oo 2.02 Y o.99-

Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys Zn-acetic acid cleavage ofN-e-pyridyl-4-methyloxycarbonyl blocking group.A mixture of 5.94 mg. ofPhe- Ser-Trp-Gly-Ala-Glu-Gly-Gln-(e-i-Noc)-Lys in 0.7 ml. of 50% aceticacid and 180 mg. of zinc dust is stirred at 25 for 1 hr. The zinc isremoved by centrifugation. One-half of the reaction is charged on a 2.5cm. x 100 cm. column of Sephadex G-50 (fine). The flow rate is 8.5BIL/20 min., separation is followed by a UV. monitor at 280 nm., andfractions 46-49 are combined, concentrated in vacuo and freeze-dried:1.43 mg. of Phe-Ser-Trp-Gly-Ala- Glu-Gly-Gln-Lys is obtained. It shows asingle zone by TLC in chloroform-methanol-water (40:47: 13), byhypochlorite, Ehrlich, and ninhydrin reagents.

EXAMPLE IV Preparation of glutamyl-glutamyl-lysyl-seryl-alaninet-Boc-Ala-Resin (6.5 g., 5 mm.) is treated in the following manner: (a)wash the amino acid resin 3 times with 45 ml. of methylene chloride; (b)wash 3 times with 45 ml. ethanol; wash 3 times with 45 ml. of aceticacid; ((1) remove the terminal t-Boc protecting group by treating theamino acid resin material with anhydrous hydrogen chloride in aceticacid for 30 minutes; (e) wash the deblocked Ala-Resin 3 times withacetic acid; (f) wash 3 times with ethanol; (g) wash 3 times withmethylene chloride; (h) neutralize the Ala-Resin by treating it with 40ml. of a solution of triethylamine in methylene chloride (1:9) for 10minutes; (i) wash times with methylene chloride; (i) add 3.7 g. (12.5mm.) of t-Boc- O-Bz-Serine in 30 ml. methylene chloride and stir forminutes; (k) couple by adding 12.5 mml. of N,N'-dicyclohexylcarbodiimidein methylene chloride to the reaction and allow it to stir for 2 hours;(m) wash the t-Boc-O-Bz- Ser-Ala-Resin 3 times with methylene chlorideand 3 times with ethanol. By repeating the above operations andemploying the following amino acids, cc-BOC-e-i-NOC- lysine 4.9 g. (12.5mm.), a-Boc-y-Bz-glutamic acid 4.3 g. (12.5 mm.), andu-Boc-y-Bz-glutamic acid 4.3 g. (12.5 mm.), 10.9 g. of the pentapeptideBoe-Glu-Glu-Lys-Ser-Ala-Resln Bz Bz -Noc 0132 is obtained.

Removal of the pentapeptide from the resin is accomplished by cooling amixture of 2.2 g. (1 mm.) of the above peptide-Resin product in 3 ml. ofveratrole to 0.5

C. and adding 10 ml. of trifiuoroacetic acid. Then 10 ml. of hydrogenfluoride is condensed into the reaction mixture using an acetone-Dry Icebath. The cooling bath is removed and the reaction is stirred at roomtemperature for 1 hour. The hydrogen fluoride and trifluoroacetic acidare removed by passing a stream of nitrogen through the reaction withstirring. The residue is treated 2 times with 25 ml. of 5% aqueousacetic acid. The resin is removed by filtration and washed 3 times with15 ml. of ethyl ether. The aqueous filtrate is washed 2 times with 15ml. of ether. The aqueous portion is freeze-dried twice to atford 700mg. of Glu-Glu-(e-i-Noc)-Lys-Ser-Ala.

The epsilon blocking group of lysine is removed by mixing 10 mg. ofGlu-Glu-(e-i-Noc)-Lys-Ser-Ala and mg. of zinc dust in 1 ml. of 50%aqueous acetic acid at room temperature for 1 /2 hours. TLC indicatedcomplete removal of i-Noc blocking group.

The t-Boc-Ala-Resin starting material for the solid phase peptidesynthesis in the above examples is prepared in the following manner: 50g. (92.5 meq. Cl) chloromethylated polystyrene resin with 1%crosslinking and 1.85 meq. Cl/gm. (Bio-Beads S-Xl 200400 MeshChloromethylated available from Bio-Rad Laboratories) and 17.5 g. (92.5meq.) of t-Boc-Ala is added to 350 ml. peroxide free tetrahydrofuran.The reaction is stirred under nitrogen and 8.4 gm. (83.3 meq.)triethylamine is added in 50 ml. of tetrahydrofuran. The reaction isheated at reflux in an oil bath for 65 hours. The reaction is cooled toroom temperature and filtered. The solid material is washed 2X 250 ml.ethanol, 2x 250 ml. water, 2x 250 ml. methanol, and 2X 500 ml. methylenechloride and dried in vacuo at room temperature to yield 55.7 gm. t-Boc-Ala-Resin which contains 0.770 meq. of t-Boc-Ala/ gm. of solid.

What is claimed is:

1. In a process for the synthesis of peptides built up from amino acidsby condensing, in a series of standard peptide condensation reactions,members of the group consisting of amino acids and peptides built upfrom amino acids, wherein at least one of said members is or containslysine, the improvement which comprises protecting the epsilon aminogroup of lysine during the condensation reactions with apyridyl-4-methyloxycarbonyl group.

2. In a process for the synthesis of peptides built up from amino acidsby condensing, in a series of standard peptide condensation reactions,members of the group consisting of amino acids and peptides built upfrom amino acids, wherein at least one of said members is or containslysine, the improvement which comprises protecting the epsilon aminogroup of lysine during the condensatlon reactions with apyridyl-4-methyloxycarbonyl group and removal of said group by zinc dustin the presence of an acid.

3. The process of claim 2 wherein the acid employed in removing thepyridyl-4 methyloxycarbonyl blocking group is acetic acid.

4. The process of claim 2 wherein from 10 to 200 parts by weight of zincdust is employed per part by weight of pyridyl-4-methyloxycarbonyllysinecontaining compound.

References Cited UNITED STATES PATENTS 3,655,636 4/1972 Young 260-1125LEWIS GOTTS, Primary Examiner R. J. SUYAT, Assistant Examiner US. Cl.X.R. 260-295 D

